JPH10512235A - IGF / IGFBP complex for promoting bone formation and regulating bone remodeling - Google Patents

Abstract

  (57) [Summary] Administer IGF and IGFBP to treat bone marrow disease, connective tissue disease, drugs, pregnancy, lactation, chronic hypophosphatemia, hyperphosphatasia, insulin-dependent diabetes, anorexia nervosa, cadmium poisoning, juvenile osteoporosis, Stimulate new bone formation in subjects with bone loss due to Paget's disease, osteoarthritis and periodontal disease. IGF-I and IGFBP-3 are optionally combined with an agent that inhibits bone resorption.

Description

DETAILED DESCRIPTION OF THE INVENTION         IGF / IGFBP complex for promoting bone formation and regulating bone remodeling Technical field   The present invention relates generally to polypeptide factors and their use in bone growth and maturation. About. Specifically, the present invention provides for the presence or absence of an inhibitor of bone resorption. -Like growth factor (IGF) and insulin-like growth factor binding protein in the presence (IGFBP) for use in stimulating bone formation.Background art   Growth factors are responsible for a wide variety of biological responses (eg, DNA synthesis, cell division, Differentiation, expression of specific genes, etc.) in a defined population of target cells Is a polypeptide. Transforming growth factor β1 (TGF-β1), TGF-β2 , TGF-β3, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblasts Including growth factor (FGF), insulin-like growth factor I (IGF-I), and IGF-II Various growth factors have been identified.   IGF-I and IGF-II are related in amino acid sequence and structure, and Has a molecular weight of about 7500 daltons. IGF-I is growth hormone And is therefore a major mediator of postnatal skeletal growth. IGF-I It is also related in the action of various other growth factors. Because like this Treatment of cells with growth factors leads to increased IGF-I production. IGF-I and IG Both F-II have insulin-like activity (hence the name), and Involved in the growth and differentiation of skeletal tissue (eg, muscle and bone) and non-skeletal tissue. Mitogenic to various types of cells that feed (stimulates cell division ).   IGF is measured in serum to determine abnormal growth-related symptoms (eg, gigantism, acromegaly, Dwarfism, various growth hormone deficiencies, etc.). IGF is in many organizations But most circulating IGF is thought to be synthesized in the liver .   Unlike most growth factors, IGF is present in the circulation in substantial amounts, Only very low fractions of GF are free in circulation or other body fluids. Big Part of the IGF is complexed with an IGF binding protein. IGF in the blood is mainly IGF Complexed with BP-3, which is the major circulating IGF binding protein. Ho Almost all IGFs are non-covalently associated, referred to as IGF-I or IGF-II, IGFBP-3. IGF-specific binding protein and acid labile subunit (Acid Labile Subunit) circulates in a ternary complex of larger proteins called unit) (ALS) . The ternary complex is composed of equimolar amounts of each of the three components. ALS is a direct IGF It has no binding activity and appears to bind only to preformed IGF / IGFBP-3 complexes. You. The triple complex of IGF + IGFBP-3 + ALS has a molecular weight of about 150,000 daltons. In the circulation, this ternary complex is composed of IGF-I and IGF-I, which prevent rapid changes in free IGF. And acts as a buffer and buffer for IGF-II. Blum , W.F. et al., "Plasma IGFBP-3 levels as clinical indicators",Modern Concepts in In sulin-Like Growth Factors , E.M. Spencer ed., Elsevier, New York, pp. 381-393. , 1991.   It is beneficial to have most of the circulating IGF in the complex. IGF is circulating glucose Excessive free IGF can cause severe hypoglycemia because it has an insulin-like effect on levels Can cause illness. In contrast to low levels of free IGF and IGFBP-3, There is a substantial pool of free ALS, which is the IGF / IGFBP-3 complex entering the circulation, Ensure immediate formation of the ternary complex.IGF Binding protein   IGFBP-3 is the most abundant IGF binding protein in the circulation. In recent years, Wood et al. (Mol . Endocrin. (1988),Two: 1176-85) and Spratt et al. (Growth Factors(1990) ,Three: 63-72) described the cloning and expression of human IGFBP-3. Its structure is It is incorporated herein by reference. The gene for IGFBP-3 contains 291 amino acids. And the first 27 represent the characteristic signal sequence. Therefore, the mature protein Contains 264 amino acids and has a predicted molecular weight of 28,749 (glycosylated or other Excluding post-translational changes). Chinese hamster with human IGFBP-3 gene -Expressed in ovarian ("CHO") cells, subjected the conditioned culture medium to SDS electrophoresis, When transferred to a nitrocellulose membrane for ligand binding analysis, Sp ratt et al., protein bands: 43-45 kd doublet, 28 kd band and And the presence of a minor 31 kd band ”(p. 69). This is a post-translational change Indicates the presence of 43-45 kd doublet by side-by-side comparison Was also found in human serum.   FIGS. 12-15 show IGFBP-3 cores suitable for use in various forms in the present invention. 1 shows the nucleotide sequence and deduced amino acid sequence.   It is not clear which tissue is the primary source of circulating IGFBP-3, but the synthesis is Shown in a number of cell types. They are human fibroblasts, liver cells ( And possibly osteoblasts). Contains IGFBP-3 cDNA CDNA libraries have been obtained from liver and other tissues. Vascular endothelium The vesicle produces IGFBP-3 and may be a major source of systemic IGFBP-3.   IGFBP-3 is purified from natural sources and produced by recombinant means. You. For example, IGFBP-3 is available from Martin and Baxter (J . Biol. Chem.(1986)261: 875 Purification from natural sources using processes such as those described in 4-60) Can be made. IGFBP-3 is also available from Sommer, A. et al.Modern Concepts of Insulin-Like Growth Factors , E.M. Spencer, Elsevier, New York, pp. 715-728, 1991. Can be synthesized by the recombinant organisms discussed. This recombinant IGFBP-3 contains one IGF-I : 1 molar stoichiometry.   At least five other different IGF binding proteins are found in various tissues and body fluids Has been identified. All of these proteins bind IGF, but these Originating from separate genes, and these have different amino acid sequences. You. Therefore, these binding proteins are not just analogs of common precursors. No. For example, Spratt et al. Compared the amino acid sequences of IGFBP-1, -2, and -3. Success 28% of the total 264 amino acids in mature protein between IGFBP-3 and IGFBP-1 And only 33% amino acid between IGFBP-3 and IGFBP-2 The acids are the same. Spratt et al. Reported that a similar part of the binding protein binds IGF. Area. Unlike IGFBP-3, other circulating IGFBPs are IGFs. Not saturated. All six known IGFBPs are available from Shimasaki and Ling,Prog . Growth Factor Res. (1991)Three: 243-66 and compared.   Spencer et al. (1991)Bone 12: 21-26; and Tobias et al., (1992)Endocrinolog y131 : 2387-2392 reports the stimulation of bone formation by IGF-I.   Rat trabecular bone is the bone-forming bone like human trabecular bone Shows the link to absorption. As a result, the increased absorption resulting from estrogen deficiency is increased. This results in added bone formation. This bone formation can be suppressed by inhibiting bone resorption. adult This association of formation and absorption in E. coli may involve a site-specific linkage of events. Established In the site-specific chain of this event, usually the same after bone resorption At the site, bone formation continues. Frost (1985)Clin . Orthop. Rel. Res. 200: 198 -225.   Bone formation is largely a requirement for mechanical support of the skeleton, without earlier bone resorption. There is also evidence that this can occur in an increasing context (modeling) . Parfitt, A.M. et al. (1984)Calcif . Tissue Int. 36: 5123-5128.   The present invention provides for the administration of an IGF / IGFBP complex, or an IGF / IGFBP complex and bone In vivo to stimulate new bone formation through the administration of drugs that inhibit resorption A single or combination therapy is provided. These combinations are Provide more effective treatments for prevention of loss and bone replacement.Disclosure of the invention   The present invention relates to the initiation and promotion of osteogenesis and the Discloses a use for adjusting bone remodeling. The IGF / IGFBP-3 complex alone or Or may be used with an inhibitor of bone resorption.   The present invention stimulates bone formation in subjects with bone marrow disease causing bone loss A method for doing so is disclosed. This method can be performed in the presence of an inhibitor of bone resorption or Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3 in the absence. Include.   In another embodiment, the invention has a connective tissue disease that causes bone loss Disclosed are methods for stimulating bone formation in a subject. This method is Administering to the subject an effective dose of the IGF-I / IGFBP-3 complex.   In another embodiment, the present invention treats a subject having drug-related osteoporosis. A method is disclosed. The method comprises administering a pharmaceutically effective dose of an IGF-I / IGFBP-3 complex Is administered to the subject.   In another embodiment, the invention relates to pregnancy, lactation, chronic hypophosphatemia, hyperphosphatemia. Tattosis, insulin-dependent diabetes, anorexia nervosa, cadmium poisoning, juvenile Disclosed is a method of stimulating bone formation in a subject having bone loss due to osteoporosis I do. The method comprises administering to a subject a pharmaceutically effective dose of an IGF-I / IGFBP-3 complex. Process.   In another embodiment, the invention provides a method for treating bone formation in a subject having periodontal bone loss. A method for stimulating is disclosed. The method comprises administering pharmaceutically effective doses of IGF-I and IGF-I. Administering GFBP-3 to the subject.   In another embodiment, the invention is directed to osteoarthritis, non-use, or reduced Stimulates bone formation in subjects with bone loss associated with prolonged exposure to force fields A method is disclosed. This method provides a pharmaceutically effective dose of IGF-I and IGFBP-3. Administering to the subject.   In another embodiment, the present invention relates to a method for inducing osteogenesis in a subject. Disclose the composition. The composition comprises IGF-I, IGFBP in a pharmaceutically acceptable excipient. -3 and bone resorption inhibitors.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the experiment used in the experiment performed to confirm the present invention (Example 2). Show the schematic of the design;   Figures 2A and 2B show the use of rhIGF-I or rhIGF1 / IGFBP-3 complex for 8 weeks. Ovariectomized (OVX) animals treated with the indicated IGF-I dose, untreated (solid bar) and Average weight (2A) and lean body mass (2B) bars compared to sham-operated controls (solid line) Show the graph;   FIG. 3 was shown using rhIGF-I or rhIGF-I / IGFBP-3 complex for 8 weeks. Ovariectomized (OVX) animals treated with IGF-I doses were treated with untreated (solid bars) and sham-operated controls. Figure 3 shows a bar graph of bone growth in cartilage compared to trawl (solid line);   4A and 4B show the use of rhIGF-I or rhIGF-I / IGFBP-3 complex for 8 weeks. Ovariectomized (OVX) animals treated with the indicated IGF-I dose, untreated (solid bar) and Bone formation rate of tibial periosteum (4A) and tibial cortex compared to sham operation control (solid line) Shows a bar graph of internal reabsorption (4B);   5A-5CC show 7.5 m complexed with sham operation (5A), OVX (5B), and rhIGFBP-3. 40 μm ground from OVX rats (5C, 5CC) treated with g / kg rhIGF-I A matching UV micrograph of a thick tibia cross section is shown (5A-5C, 3x magnification; 5CC, 5C (12 × magnification of periosteal envelope area shown), where new Severe bone formation is indicated by arrows;   Figures 6A-6D show sham-operated animals (6A), OVX animals (6B), and complexed with rhIGFBP-3. Unstained 40 μm thick sections from animals treated with 7.5 mg / kg rhIGF-I (6C, 6D) A polarized micrograph of the surface is shown (magnification 12.5x), where the outer lamellar layer is Barred and the inner lamellar layer is indicated by an arrow;   7A-7D show the results using rhIGF-I or rhIGF-I / IGFBP-3 complex for 8 weeks. (Solid bar) and fake hand of ovariectomized (OVX) animals treated with a given IGF-I dose Metaphyseal (7A), epiphyseal (7B), lumbar vertebral bodies (7C) and 5 shows a bar graph of the rate of bone formation of reticular bone in the femoral neck region (7D), where Talisks indicate p <0.05 compared to OVX group;   8A-8C show sham-operated controls (8A), OVX (8B) and 7.5 ml / kg rhIGF-I. Microscopy of reticular pillars in the distal femoral epiphysis from rats treated with the 8C / IGFBP-3 complex (8C) The picture is shown, where the arrows indicate the re-absorption holes (pits) and the tears are newly formed Showing damaged bone;   9A-9C show sham-operated control (9A) OVX (9B) and 7.5 mg / kg rhIGF-I / I. GFBP-3 complex treated rats (9C) stained by von Kossa mineral staining method. Matching micrograph (3 × magnification) of a 4 μm section collected from the isolated distal femur Show;   FIGS.10A-10C show composites with sham-operated controls (10A), OVX rats (10B) and IGFBP-3. From the mid-femoral neck region of rats (10C) treated with the combined 7.5 mg / kg rhIGF-I Shows a cross-sectional image (2.5 × magnification) of a section taken;   FIG. 11 shows the amino acid sequence of human IGF-I used in the present invention (SEQ ID NO: 1);   FIG. 12 shows the amino acid sequence of human IGFBP-3 (SEQ ID NO: 2), where Talisks indicate glycine substitution of alanine for naturally occurring heterogeneity in this sequence. You;   FIG. 13 shows human I used to produce rhIBFBP-3 for use according to the invention. Sets forth the nucleotide sequence of GFBP-3 (SEQ ID NO: 3 and SEQ ID NO: 4);   FIG. 14 shows another alternative used to produce rhIBFBP-3 for use according to the invention. Shows the nucleotide sequence (SEQ ID NO: 5); and   FIG. 15 shows the secondary used to produce rhIGFBP-3 for use according to the invention. SEQ ID NO: 6 and SEQ ID NO: 7 are shown.MODES FOR CARRYING OUT THE INVENTION A.Definition   As used in this specification and the appended claims, unless explicitly stated in context. Note that the singular forms "a," "an," and "the" include plural referents. There must be.   The term "connective tissue disease" refers to osteogenesis imperfecta, Ehlers-Danlos syndrome, Fan syndrome, cutaneous laxity, homocystinuria, Mankes syndrome and scurvy Including such symptoms. These are associated with and cause bone loss Have been.   "Drug-related osteoporosis" refers to osteoporosis, where the only cause identified is a drug I do. This definition includes all known drugs that cause osteoporosis, and osteoporosis Includes drugs that are later discovered to cause the disease. Can cause osteoporosis Examples of drugs known to include corticosteroids, heparin, oral anticoagulants , Anticonvulsants, methotrexate, thyroid hormone, lithium and gonadotropy Release analogues.   "Insulin-like growth factor (IGF)" includes but is not limited to IGF-I and IGF-II Includes a family of factors that are not performed. IGF has a molecular weight of about 7500 daltons. It is a repeptide. IGFs can be obtained from natural sources or by recombinant means. Can be prepared. Preferably, the IGF is IGF-I from a human source. Most preferred In particular, the IGF is IGF-I from a human source. Most preferably, the IGF is used herein. Produced by recombinant means, such as by the method detailed in Example 3 of Human IGF-I called rhIGF-I.   "Insulin-like growth factor binding protein (IGFBP)" is IGFBP-1, IGFBP-2, IGFBP Binding agents, including but not limited to BP-3, IGFBP-4, IGFBP-5 and IGFBP-6 Includes protein families. Each of the IGFBPs can be obtained from natural sources, or Alternatively, it can be prepared by recombinant means as detailed in Example 4 herein. IG At least one form of FBP (eg, IGFBP-3) is known as IGF and and ALS Complexes with a third molecule. IGFBP is also a mixture of any combination of the six IGFBPs. It can be a compound. Such a mixture may be different for IGF-I and / or IGF-II. Binding affinity, the ability of some IGFBPs to bind to the cell surface, and different halves Period is available.   As used herein, a "therapeutic composition" comprises an IGF and a bone resorption inhibitor. And IGF-binding protein (IGFBP). The therapeutic composition can also include May contain excipients such as water, minerals, and carriers such as proteins .   The method of the present invention comprises administering to a subject in need of IGFBP replacement a bone resorption inhibitor. Local bone repair or treatment in combination with IGF with or without Intend. The IGF can be of any IGF family, including IGF-I and IGF-II, or Including, but not limited to, these combinations. IGF-I and IGF-II combined If so, the ratio of IGF-I to IGF-II ranges from 0.01: 1 to 99: 1.   "IGFBP-1" has the molecular structure of Brewer et al.Biochem . Biophys. Res. Comm. ( 1988)152(3): 1289-1297, and published on September 21, 1989, to Drop et al. IGF binding protein disclosed in PCT Publication No. WO 89/98667 and The specification is incorporated by reference. Human IGFBP-1 has 234 amino acids and approximately 28 kd. Has a molecular weight.   "IGFBP-2" contains 289 amino acids (human) and has a distribution of 36 kd under non-reducing conditions. Has child weight. The amino acid sequence of human IGFBP-2 isEMBO J.(1989)8: 2 493-2502 determined from cDNA clones isolated from human fetal liver library. And is incorporated herein by reference. IGFBP-2 also binds to cell surfaces. Can match. IGFBP-2 has selectivity for IGF-II and therefore contains IGF-II. It is suitable for the formulation which is used.   “IGFBP-3” is a suitable IGFBP in an IGF / IGFBP complex. Natural and recombinant IGF BP-3, and some N- and C-terminal fragments, are IGF-I and IGF-II To join. Human IGFBP-3 contains 264 amino acids and has three possible N-links It has a glycosylation site. IGFBP-3 is the major IGFBP in blood.   Almost all IGF-I or IGF-II in the blood binds to IGFBP-3. IGF / The IGFBP-3 complex is usually in complex form in humans and other mammals and birds. Circulates in This complex forms a third protein that is present above the concentration of IGF and IGFBP-3. It is associated with protein (ALS) quality. Thus, in circulation, ALS is an IGF / IGFBP-3 complex And is found both in free form. The resulting triple complex is approximately It has a size of 150kD. IGF, derived from either natural or recombinant sources Administration of the preformed complex with IGFBP-3 usually results in excess complex with ALS in triplicate. Causes formation. This type of treatment involves circulating IGFs gradually released from the ternary complex It appears to produce long-term increases in levels. This mode of administration is based on free IGF-I Adverse side effects with administration, such as hypoglycemia, growth hormone suppression and AL Avoid S production, as well as release of endogenous IGF-II. Because the administered exogenous Free IGF-I replaces endogenous IGF-II in the normal circulating IGF-II / IGFBP-3 complex. It is because it changes.   "IGFBP-4" and "IGFBP-6" are glucosylated proteins widely distributed in the body. Quality. The primary structure of IGFBP-4 is Shimasaki et al.Mol . Endocrinol.(1990)Four:14 51-1458 (incorporated herein by reference). IGFBP-6 is cDNA is Shimasaki et al., (Mol . Endocrinol.(1991)Five: 938-48) And has significantly greater affinity for IGF-II than for IGF-I, and It may be suitable for formulations containing GF-II. This document is incorporated herein by reference. Is done.   "IGFBP-5" is a non-glycosylated 252-amino acid protein . Shimasaki et al. (J . Biol. Chem.(1991)266: 10646-53) is a human placenta library -Derived human IGFBP-5 cDNA was cloned (hereby incorporated by reference). .   The binding, metabolic and pharmacokinetic properties required for IGF / IGFBP complex formulations Depending on, these binding proteins may be added to the complex formulation in various proportions. You. These IGFBPs are combined with IGF-I and / or IGF-II in various ratios. obtain.   The term "inhibition of bone resorption" refers to a direct or indirect modification of osteoclast formation or metabolism. Prevention of bone loss due to, in particular, either the inorganic phase and / or the organic matrix phase Refers to the inhibition of removal of existing bone. Accordingly, the term "bone remodeling" as used herein "Inhibitors of resorption" may be due to direct or indirect alterations in osteoclastogenesis or metabolism A drug that prevents bone loss.   The term "bone remodeling" renews the skeleton, and microdamage loses skeletal integrity The process of repairing microdamage before it accumulates to a point. Most humans Metabolic bone disease in adults results from disruptions in the remodeling process. Individual reshaped packets (packet) is known as the "Basic Multicellular Unit" (BMU). The remodeling that occurs in BMU Follow a pre-programmed order: activation → resorption → formation (ARF). (Recker, R.R ., "Bone Histomorphometry: Techniques and Interpretation." Boca Raton, FL, 1983, pp. 37-57).   “Osteogenesis” refers to the number and / or activity of BMUs (basal multicellular units) that produce new bone. Includes increased sex. This process can take place anywhere in the skeleton, and Reticulated, regardless of the functional differences and turnover rates that bone sites may have in the skeleton It can affect bone and / or cortical bone. (Frost (1986)Intermediary Organization of the Skeleton , Volumes I and II , CRC Press, Boca Raton, FL.).   The term “effective for bone formation” refers to the formation and differentiation of mature bone or early osteoprogenitor cells Means the amount that affects As used herein, an osteogenically effective dose may also be referred to as Effective ".   The term "subject" as used herein is in need of treatment, i.e., bone repair or A living vertebrate, such as a mammal or bird, in need of bone replacement. like that Need for osteogenesis imperfecta, Ehlers-Danlos syndrome, Marfan syndrome, skin Connective tissue diseases such as flaccidity, homocystinuria, Menkes syndrome and scurvy ; Corticosteroids, heparin, oral anticoagulants, anticonvulsants, methotrexate Drugs such as thyroid hormone, lithium and gonadotropin releasing analogs Pregnancy; lactation; chronic hypophosphatemia; hyperphosphatasia; Surin-dependent diabetes mellitus; Anorexia nervosa; Paget's disease of bone; Cadmium poisoning; Teeth Periodontal disease; osteoarthritis, prolonged hospitalization, muscular bone as occurs during paralysis or hemiplegia Nonuse of case system; and prolonged exposure to low gravity fields.   As used herein, the term “treatment” is (1) used prophylactically to prevent the progression of bone loss. Providing the subject with an amount of the substance that is sufficient for use; or (2) Provide the subject with sufficient quantities of the substance to reduce or eliminate the problem Means B.General method   Drugs that prevent bone loss and / or regenerate lost bone Has been evaluated using This animal model is well established in the art. (E.g., Wronski et al. (1985)Calcif . Tissue Int. 37: 324-328; Kimmel et al. ( 1990)Calcif . Tissue Int. 46: 101-110; Durbridge et al. (1990)Calcif . Tissue In t.  47: 383-387; and Miller et al. (1991)Bone 12: 439-446; these references are , Incorporated herein in their entirety). Wronski et al. Describes the relationship between bone loss and bone remodeling inCalcif . Tissue Int.  43: 179-183).   Examples of bone resorption inhibitors include estrogens, such as conjugated estrogens, Tamoxifen, bisphosphonate, calcitonin, or prevent bone resorption Other small peptides or molecules that may be harmful are included. (Turner et al. (1987)J . Bone M ineral Res.  2: 115-122; Wronski et al. (1988)Endocrinology 128: 681-686; And Wronski et al. (1989)Endocrinology 125: 810-816; Pfeilshifter et al. (1987)P roc . Natl. Acad. Sci. USA  84: 2024-2028; Turner et al. (1988)Endocrinolog y  122: 1146-1150). An example of a small peptide is echistatin, Arginine-glycine is recognized by several cell surface adhesion receptors Contains a syn-aspartic acid (RGD) sequence and reveals osteoclast interactions Destroy (Fisher et al. (1993)Endocrinology 132: 1411-13). Another of bone resorption factor Examples are OPF or osteoclastogenic factor (PCT Publication No.WO 93/01827 February 4, 1993 Published). Other drugs under study or proposed to inhibit bone resorption Is mitramycin, gallium nitrate, glucocorticoid, transformin Growth factor beta (TGF-beta), interferon gamma and amylin. (Zaidi et al. (1 (October 992)Curr . Opin. Therapeutic Patents, 1517-38).   The entire molecule of a particular inhibitor may be used, or if necessary, the inhibitor. Only the functional part of the bitter molecule may be used. Bisphosphonates are pamidrone Acid, alendronate, tildronate, risedronate, and other fruits Test compounds, but are not limited thereto.   Certain growth factors can inhibit bone resorption or are anti-resorbable. That Examples of such growth factors include but are not limited to transforming growth factor beta Not done.   According to the method of the present invention, a preparation containing a complex of IGF and IGFBP is converted into a bone resorption inhibitor. Administered with or without tar.   Preferably, the IGF is IGF-I, although IGF-II can also be used. Preferred Alternatively, the IGFBP is IGFBP-3. IGF and IGFBP-3 naturally bind in a 1: 1 molar ratio Therefore, an equimolar amount composition of IGF and IGFBP-3 is preferred. Nevertheless, Can be formulated with an IGF: IGFBP-3 molar ratio ranging from 0.5: 1 to 1.5: 1. further Preferably, the molar ratio is from 0.9: 1 to 1.3: 1; and most preferably, the set The composition is formulated in a molar ratio of about 1: 1.   According to the method of the present invention, IGF and IGFBP-3 are obtained from natural or recombinant sources. The obtained human protein. Most preferably, IGF and IGFBP-3 are recombinant Human IGF- made by means and named rhIGF-I and rhIGFBP-3, respectively I and human IGFBP-3. rhIGFBP-3 is glycosylated or non-glycosylated Can be a type. E. E. coli is a source of non-glycosylated IGFBP-3. Glycosi Luminated IGFBP-3 can be obtained from CHO cells.   The method of the present invention makes it possible to formulate the conjugate in a manner readily apparent to those skilled in the art. provide. Preferably, the IGF and IGFBP-3 are administered prior to administration to the treated individual. Complexed. Preferably, the complex comprises normal saline or phosphate Almost equimolar, dissolved in a physiologically compatible carrier, such as buffered saline. Formed by mixing a small amount of IGF-I and IGFBP-3. Most preferably, rh Sufficient enough to form an equimolar complex between the concentrated solution of IGF-I and the concentrated solution of IGFBP-3. Time, mixed together.   Pharmaceutical composition of the invention containing IGF, IGFBP and bone resorption inhibitor for administration The substance, in addition to pharmaceutically acceptable excipients, is used for osteogenesis to promote osteogenesis. It contains an effective amount of IGF and IGFBP, and an inhibitory amount of a bone resorption inhibitor. Suitable excipients include most carriers approved for parenteral administration Water, saline, Ringer's solution, Hank's solution, and glucose, Cactose, dextrose, ethanol, glycerol, albumin solution, plasma , A solution containing other proteins, and the like. These compositions are needed Depending on stabilizers, antioxidants, antibiotics, preservatives, buffers, surfactants, and Other auxiliary additives may be included. IGF / IGFBP complex and bone resorption inhibitor Can also be delivered in a slow release from a suitable carrier.   A variety of vehicles can be used with the present invention. A suitable vehicle for parenteral administration For a complete discussion of Kullu, see E.W.Martin's `` Remington's Pharmaceutical Sciences '' (Ma ck Pub. Co., latest edition). Related to excipient vehicle and formulation Sections are incorporated herein by reference. Such formulations are generally It is known to those skilled in the art and is administered systematically to provide systemic treatment.   The IGF / IGFBP complex and the agent that inhibits bone resorption can be administered as a single composition to the subject. And may be administered simultaneously or continuously. If administered continuously, IG The period between F / IGFBP complex administration and bone resorption inhibitor administration is typically It is one day to one year, and preferably one week to six months. IGF / IGFBP When the complex and the agent that inhibits bone resorption are administered as a single composition, The molar ratio of the IGF / IGFBP complex to the bone resorption inhibitor It varies considerably depending on the type of protein and the formulation of the IGF / IGFBP complex. Hot The ratio for most compounds is between about 100: 1 and about 1: 100. In addition, a single composition When When administered as a composition, the IGF / IGFBP complex and the bone resorption inhibitor Can be administered as separate molecules, or each molecule is well known to those of skill in the art. They can be joined or fused according to techniques.   The exact dose will depend on the subject's age, size, sex, condition, and the nature of the disorder being treated. It changes as an inevitable result such as severity. Thus, the exact effective amount in advance It cannot be specified and must be determined by the care provider. But the right amount It can be determined by routine experimentation using the animal models described below.   Generally speaking, for systemic treatment, an effective dose of the IGF / IGFBP complex is from about 1 μg to about 10 μg. The range is mg IGF / kg body weight. Effective dose of bone resorption inhibitor selected for administration Depends on the particular inhibitor given. For example, the effective dose of estrogen is about 0. 25-1.5 mg / day. Effective doses of bisphosphonates vary, but are generally about 0.0 Between 5 μg / kg body weight and about 15 mg / kg body weight. An effective dose of calcitonin is about 0.05  IU (International Units or Medical Research Council Units) / kg body weight ~ About 2.5 IU / kg body weight.   Effective amounts for topical administration range from about 0.01 μg to 1 mg IGF / IGFBP complex.   The methods and compositions of the present invention are useful for treating bone fractures, bone defects, and weak bones. It is useful for treating. These diseases include plasmacytopathy, leukemia, phosphorus Bones such as pamas, systemic mastocytosis, anemia, lipidosis, and mucopolysaccharidosis Medullary disorders; osteogenesis imperfecta, Ehlers-Danlos syndrome, Marfan syndrome, skin Connective tissue diseases such as flaccidity, homocystinuria, Mankes syndrome, and scurvy Patients: corticosteroids, heparin, oral anticoagulants, anticonvulsants, methotrexe Drugs such as thyroid hormone, lithium and gonadotropin releasing analogs Pregnancy; lactation; chronic hypophosphatemia; hyperphosphatasia; Insulin-dependent diabetes mellitus; anorexia nervosa; Paget's disease of bone; juvenile osteoporosis; Cadmium poisoning; periodontal disease; and osteoarthritis. Further, the method of the present invention and The composition results from prolonged resting bed or exposure to reduced gravitational fields It is useful for treating weak bones resulting from such musculoskeletal disuse.   According to one method of use, the IGF / IGFBP complex may be used locally for bone growth or repair. Concomitant administration with bone resorption inhibitors at the specific area required, or Is administered locally, either with administration of a bone resorption inhibitor in a separate vehicle obtain. Thus, IGF / IGFBP complexes and / or bone resorption inhibitors are For example, treated by injection or surgical implantation in a sustained-release carrier It can be implanted directly at the site to be affected. Suitable carriers include hydrogels, controlled or Rix. Currently, the preferred carrier is hydroxyapatite phosphate Tricalcium (available from HA-TCP, Zimmer, Inc., Warsaw, IN) or similar (available from ollagen Corporation, Palo Alto, California) Atelopeptide cola containing specific particulate calcium phosphate mineral components It is a preparation of gen. IGF / IGFBP complex in collagen / mineral mixture implant And / or administering a transplant composition containing a bone resorption inhibitor. Currently preferred.   The IGF / IGFBP complex and / or bone resorption delivered in a sustained-release vehicle Inhibitors may also be used to improve implant fixation, for example, in joint reconstruction. Improves ingrowth of new bone into genital prostheses and dental or orthopedic implants Especially useful for. Alternatively, the IGF / IGFBP complex is delivered in a separate vehicle Inhibitors that can be delivered into an implant and vice versa.   Dental and orthopedic implants are combined with bone resorption inhibitors to To increase the adhesion of the implanted device, it can be coated with an IGF / IGFBP complex. Ah Alternatively, IGF / IGFBP complexes can be used to coat implants and The absorption inhibitor can be administered concomitantly and in a separate vehicle, and The reverse is also true.   In general, the implant device is an IGF / IGFBP complex and / or bone resorption as follows: It can be coated with a yield inhibitor. IGF / IGFBP complex (and bone resorption, if desired) Phosphate inhibitor buffered saline containing 2 mg / ml serum albumin. Dissolve in water (PBS) at concentrations ranging from 0.01 mg / ml to 200 mg / ml. The porous end of the implant Immerse in this solution and air-dry (or freeze-dry) or Immediate transplant. If desired, reduce the viscosity of the coating solution to a final concentration of 0.1 mg / Addition of hyaluronate at ml-100mg / ml or other pharmaceutically acceptable excipients Is increased by the addition of Alternatively, the IGF / IGFBP complex-containing solution ((and Bone resorption inhibitor), collagen gel or human collagen (e.g., Mix at a lagen concentration of 2 mg / ml to 100 mg / ml to form a paste or gel, then It is used to coat the porous end of the implant device. Coated transfer Implantation devices should be used to minimize soft tissue ingrowth into the implantation site while minimizing Immediately place at the bone site or air dry before transplantation to maximize new bone formation It is returned to water again with PBS. C.Example   The following examples illustrate the extraction, isolation, formulation and use of the compositions and methods of the present invention. A full disclosure and description of how to do this is provided to provide those skilled in the art with It is not intended to limit the scope of what is considered to be its invention. Numbers used Efforts have been made to ensure accuracy with respect to (eg, amount, time, temperature, etc.) Some experimental errors and deviations should be considered. Unless otherwise indicated, Parts are parts by weight, temperature is in degrees Celsius, pressure is at or near atmospheric. , And other parameters are conventional and generally recognized by those skilled in the art. Follow the meter.                                 Example 1   This example demonstrates free IGF-I and IGF in female rats with ovariectomy-induced osteoporosis. 2 illustrates the use of -I / IGFBP-3 complex. In this experiment, rats were treated with human recombinant IGF-I and IGF-I. Treated with GFBP-3. rhIGF-I (Ciba-Geigy) is synthesized in yeast and sterile water And stored at -70 ° C. rhIGFBP-3 was prepared using the procedure described in Example 4. In order (Celtrix Pharmaceuticals, Inc., Santa Clara, CA) Was dissolved in phosphate buffered saline and stored at -70 ° C until use. Before dosing First, thaw these solutions and mix sufficient amounts of IGF-I and IGFBP-3, etc. Molar amounts of the two proteins were provided. This experiment was performed in an ovariectomized rat. Of IGF-I / IGFBP-3 complex to increase spongy weight and other parameters Demonstrate.   In this example, a young female rat weighing 90-100 g was ovariectomized by the dorsal route. And divided into six groups of eight rats each. Another group consisted of eight intact It consisted of sham-operated control rats of the same age. Six weeks after ovariectomy, Vehicle or one of the following combinations of IGF-I and IGFBP-3 or IGF alone Rats were treated as indicated, alone. In these experiments, the IGF and And the amount of IGFBP was calculated to provide a 1: 1 molar ratio of IGF: IGFBP:   Group 1: Sham operated controls; vehicle   Group 2: Ovariectomized controls; vehicle   Group 3: Ovariectomy; IGF-I complexed with IGFBP-3 (9.5 mg / kg) (2.5 mg / kg) kg)   Group 4: Ovariectomy; IGF-I (0.25 m) complexed with IGFBP-3 (0.95 mg / kg) g / kg)   Group 5: Ovariectomy; IGF-I (0.02 mg / kg) complexed with IGFBP-3 (0.095 mg / kg) 5mg / kg)   Group 6: Ovariectomy; IGF-I (2.5 mg / kg)   Group 7: Ovariectomy; IGF-I (0.25 mg / kg)   The complex was dissolved in an equimolar amount of IGFBP-3 (phosphate buffered saline (PBS), pH 6.0 ) And IGF-I (dissolved in 10 mm sodium acetate, pH 5.5) Form by mixing in a volume and incubating the mixture at 4 ° C. overnight. I let it. The mixture was then washed with PBS containing 0.1% rat serum albumin (pH 6.0). ). Dispense solution into aliquots containing the required amount of material per day And stored at -70 ° C until needed. Controls include dilution buffer Was administered.   Rats were treated for 22 days. Test substance was administered once daily by subcutaneous injection for one week 6 doses. One day prior to treatment and on day 17 of treatment, a 20 mg / kg In was given by intraperitoneal injection. Calcein and tetracycline are bone lime Marker for bone formation and is used to assess the amount of bone formation between doses. You. Similarly, on day 10, 20 mg / kg of demeclocycline was administered. Day 23, Twenty-four hours after the subsequent injection, the rats were sacrificed by anesthesia with carbon dioxide.   Body weight was recorded throughout the experiment. At necropsy, 0.1 ml of blood was measured for blood glucose. Collected for determination. Serum was prepared from the remaining blood and total serum IGF-I levels Was measured by radioimmunoassay (RIA). Gastrocnemius, fat around the uterus, And the uterus were removed, dissociated from connective tissue, and weighed.   Spontaneous and cortical amounts were determined by Gunness-Hey ((1984)Metab.Bone Dis. & Rel.Res. Five : 177-81), which is incorporated herein by reference. Simple Simply put, the femur is cut in half at the midshaft using a dental saw. Was. The proximal half was discarded. Dissect the distal half epiphysis using a scalpel and Was divided into sagittal halves. The bone marrow was washed with water. Dental cleated metaphyseal Sponge was dug out of both cortical hulls, combined, and 5% trichloroacetic acid ( TCA). The two pieces of the remaining cortex are also put together and another chi together with 5% TCA Put in a tube. After allowing this preparation to stand at room temperature for 16 hours, the TCA extract was subjected to atomic absorption. Used for determination of calcium by light spectroscopy. Remaining decalcified matrices The residue was washed sequentially with ethanol and methylene chloride and dried under reduced pressure. . After measuring the dry weight, the matrix was hydrolyzed with 6M HCl at 120 ° C for 5 hours. Was. In a hydrolyzate, hydroxyproline was assayed in a standard colorimetric assay (Jamall et al. (1981). )Analyt.Biochem. 112: 70-75).   The results of this experiment are shown in detail in Tables 1 and 2 and are summarized below.   Ovariectomized (Ovx in the table) control rats were replaced with sham-operated Significant difference between cortical weight and hydroxyproline when compared to control animals Was not observed (Table 2). However, cortical calcium does not Significantly increased (7%). This increase is due to the increased vertical growth of bone. And increased organ size. In ovariectomized rats, spongy weight, Calcium and hydroxyproline were reduced by 65% (Table 1).   In ovariectomized rats treated with IGF-I alone, both doses (2.5 mg / kg and And 0.25 mg / kg) increased spongy mass; however, lower doses It was more effective. Lower doses increase various parameters by 79% to 118% High doses, on the other hand, increased various parameters by 57% to 67%. Other para There was a slight change in the meter.   Ovariectomized rats treated with the IGF-I / IGFBP-3 complex were treated with various spongy proteins. It showed an increase in the parameters (Table 1). Spongy calcium, dry weight, and A significant increase in droxyproline was due to a 12 mg / kg IGF-I / IGFBP-3 dose (2.5 mg / kg IGF -I and 9.5 mg / kg IGFBP-3). The dry weight of the cortical matrix is also , 12 mg / kg IGF-I / IGFBP-3 complex significantly increased (Table 2).                                Example 2   13-week-old female Sprague Dawley rats (Charles River Associates) were ovariectomized. They were housed for approximately 3 weeks before undergoing either outing (OVX) or sham operation (Sham). You All OVXs were successful as measured by uterine weight measured at the time of sacrifice. Was. Eight weeks after surgery (assuming day 0), the rats are exposed to bone mineral density (vertebral L3-L6). Dual Energy X-ray Absorption Spectroscopy (Dual Energy X-ray Absorptometry) (DEXA) Spine scan and assigned to group ( See below). Eliminates "outsiders" and similar mean formulas between different groups (Opine) An attempt was made to have DMD values and body weight.   rhIGF-I / IGFBP-3 is prepared by mixing equimolar amounts of rhIGF-I and rhIGFBP-3. Prepared. The vehicle used for both rhIGF-I and rhIGF-I / IGFBP-3 was Phosphate buffered saline (PBS) pH.6.0.   Treatment (daily subcutaneous injection) was initiated the day after the baseline DEXA spine measurement (Day 1). Started and lasted 56 days. Two separate "pre-treatment" groups of rats (OVX and sham Surgery) was sacrificed on day 0 to provide baseline data.   Rats were divided into seven groups according to the experimental example illustrated in FIG.   (1) Sham-operated rats treated with saline ("sham control"; n = 9).   (2) OVX rats treated with saline ("OVX control"; n = 11).   (3) OVX rats treated with 0.9 mg / kg / day rhIGF-I (n = 7).   (4) OVX rats treated with 2.6 mg / kg / day rhIGF-I (n = 7).   (5) OVX rats (n) treated with 0.9 mg / kg / day of rhIGF-I in an equimolar ratio to IGFBP-3 = 8).   (6) OVX rats treated with 2.6 mg / kg / day rhIGF-I in equimolar ratio to IGFBP-3 (n = 8).   (7) OVX rats treated with rhIGF-I at an equimolar ratio of 7.5 mg / kg / day (n = 8). a.Serum measurement   On day 22, food was removed from the rats at approximately 7 am and 150 μl of blood Obtained from the tail of each rat under isoflurane anesthesia between 10 o'clock and noon. To collect blood Immediately after each rat received a normal daily treatment injection and a second blood sample Was obtained exactly 2 hours after injection. Food was returned to the rat only after this second exsanguination. Was. Plasma glucose levels were measured by a standard colorimetric assay (Table 3) . This assay uses standard procedures well known in the art, By peroxide generated from glucose as a result of treatment with oxidase The oxidation of 0-dianisidine.   During the seventh week of the study, blood was re-obtained from the tail during a regular DEXA scan. These samples were subjected to affinity chromatography procedures, specifically "GL Elution using YCO-TEK ”assay kit (Helena Laboratories, Beaumont, TX) Hemoglobin was detected at 405 nm by their absorbance and glycosylated. Hemoglobin levels were analyzed.   Sera obtained at the time of sacrifice were analyzed for total IGF-I, rhIGF-I, and rhIGFBP-3 levels. And analyzed. Serum IGF-I levels were measured by two separate assays. each The assay was performed once for all sets of samples.   First, using the Nichols Institute RIA procedure, endogenous rat IGF-I and injected The total concentration of rhIGF-I was determined. 80 μl of sample was added to 900 μl of 87.5% 2N HCl / Extract with 12.5% ethanol, centrifuge, then 200 μl of supernatant was added to 100 μl of 855 mM Neutralized with ris buffer pH 11.0. Keep the neutralized extract at -20 ° C for 1 hour and centrifuge Separate, then dilute 31-fold with phosphate buffered saline (PBS), then add 50 μl RI Analyzed by A. Hypophysectomized rats by preliminary assay (n = 6) The serum obtained from was shown to give values below the limit of detection (66 ng / ml), And this assay gives a linear response when the volume of serum varies from 10 to 100 μl. I got it.   The second assay was the serum level of rhIGF-I by immunoradiation assay (IRMA). Was quantified. This assay follows the protocol provided by the manufacturer, Sample size was 10 μl. 555 ng / ml according to the Nichols Institute RIA procedure The pooled rat serum sample containing IGF-I was tested for the detection limit of this assay. (50 ng / ml). In addition, rhIGFBP-3 for 10 μl of sample Was measured by RIA. for both rhIGF-I and rhIGFBP-3 Assay kits and procedures are available from Diagnostic Systems Laboratories (Webster, TX) Obtained from.   Serum IGF-I and IGFBP-3 levels and glycosylated hemoglobin in blood The values are shown in Table 4. b.Bone measurement   Active mineralization in all animals included in this study ) Is treated with the fluorescent bone markers decromycin and calcein ( calcein), these markers were used 9 days and 2 days before sacrifice, respectively. Was labeled by injection.   Rats were sacrificed by exsanguination under ketamine / xylazine anesthesia. Many Soft tissues were collected for histological analysis, and some of these tissues Tissue hypertrophy was examined by weighing. Long bones (tibia and femur), spine, and The mandible is detailed below for DEXA, mechanical analysis, and bone histomorphology analysis. Fixed or frozen by the following procedure.                              1.DEXA Measurement.   Spinal DEXA scan on days 0 (above), days 14, 28, 42, and 56 (The day before slaughter). Measure body composition (lean body mass and fat mass) DEXA full-body scan (excluding head and tail) to be performed on days 7, 21, 35 And on day 49.   Perform DEXA measurements using a Hologic QDR-1000 / W instrument according to the manufacturer's instructions. I went there. Rats are exposed to a gaseous anesthetic of 2% isoflurane in oxygen during the scan And the rats were also weighed while anesthetized.   The parameters measured for the tibia and femur during the DEXA analysis were long Size (mm), projected area ("total area") (cm<sup>Two</sup>), Total bone mineral content (BMC) (mg), whole spinal cord Density (BMD) (mg / cm<sup>Two</sup>), Apparent density of total bone mineral (total BMAD = BMC divided by protrusion area) To the power of 1.5) (mg / cm<sup>Three</sup>), Metaphyseal BMD (met BMD) (mg / cm<sup>Two</sup>), Cortical BMD (mg / cm<sup>Two</sup>), And epiphyseal BMD (epiph BMD) (mg / cm<sup>Two</sup>). The results are shown in Table 5 (tibia) and It is shown in Table 6 (femur). In these tables, the numerical group names are shown in Table 1. Corresponding to the name. Groups A and B are pre-treatment baseline measurements.   DEXA bone measurements are measured by both total tibia BMC value and ash weight Was confirmed by comparing the mineral content of DEXA body composition measurements on balance To compare weight changes measured by both body weight measurement and whole body mass DEXA measurement. And confirmed by.                       2.Bone histomorphology and bone structure   Vertebral body of the right femur, tibia, and lumbar spine (Lvb; L<sub>Four</sub>-L<sub>Five</sub>) Collected by autopsy and soft Tissue was washed, fixed in 70% ethanol for 48 hours, and stepwise increased in ethanol Do not remove calcium in methyl methacrylate And embedded. Longitudinal sections of the vertebral body of the lumbar spine, Cross-section of the distal tibial shaft adjacent to the mid femoral neck and tibiofibular junction Sections were cut from a Reichert-Jung supercut microtome (Reichert-Jung, Heidelberg , Germany) to a thickness of 15 μm. Dynamic tissue with three consecutive sections Unstained for morphological analysis and the other three sections were subjected to von Kossa Stained for minerals and counterstained with toluidine blue for static histomorphology Were analyzed for statistical parameters. 100 from the mid-axial and mid-shin region of the femur μm-thick cross-sectional sections were cut with a precision low-speed bone saw (Isomet, Buehler, Lake Bluff, IL) Cut, adhere to a plastic slide, rub to a thickness of about 40μm and polish (Eco met 3, Buehler, Lake Bluff, IL) and dynamic parameters of bone did. After staining with the von Kossa method and covering with coverslips, static bone tissue The same set of slides was used for morphology. Dynamic measurement of bone under UV light At double magnification, Miller et al. (1989)Bone 7: 283-287, `` Stereo logy ”, a semi-automated software program (KSS Computer Engineers, M agna, UT). Automated static bone analysis TV microscope images The analysis was performed using an analysis system, and “Image Analysis” software (KSS Comp (uter Engineers, Magna, UT).   For the thigh and shin only, the internal structure of the spongy material and the endocortical table Regarding the interconnectivity and connectivity between the surface and the spongy web, "Nodal" and " An analysis of "star volume" was performed. These are Miller and Wronski ( 1993)Anat . Record 236 : 433-441 and Bagi and Miller (1994)Anat . Record 239  : 1-12. Bone marrow astral volume is a direct measure of spongy separation. Yes, while nodal analysis shows connectivity between nodes and struts, strut types, and sponges Represents The connection between the endothelial surface and the trabecular meshwork is cortical and cortical. Is an important determinant of bone strength that connects to one anatomical function unit. The total number of endothelial-sponge junctions is counted and the sponges and others ending unbound The percentage of sponges associated with the sponges and / or endothelial surfaces of the spores is calculated. all Measurements and derived parameters describing bone kinetics and bone structure stomorphometric Committee (Parfitt et al. (1987)J.Bone Min . Res. Two: 595-610) Performed as recommended by                     3. Bone fracture strength and internal cortical structure   The soft tissue of the left femur is washed, and the bone mineral density (BMD) and bone mineral content (B MC) was used for DEXA measurement. To test the biomechanical properties of the cortex, The proximal and distal ends of the femur should be free to move parallel to the long axis of the bone. Attached to the measured fixture. Use this fixture with the MTS 858 Bionix System (MTS System Corp., Minneapolis, MN), and the rotational force was previously described (van der M eulen, M.C.H., "Bone Strength in Young, Suspended Rats", Stanford Univ. ersity Thesis, 1993). Measured (range 0-50 pounds; Model QWFK-8M / 1941, Sensotec, Columbus, O H). (Mechanical testing was conducted by the Department of Mechanical Engineering, Stanford Univ. ersity, Stanford, CA; and Rehabilitation Research and Development Cent er, Palo Alto, CA; and VA Medical Center, Palo Alto, CA). Machine After mechanical testing, the same specimen is dehydrated with increasing concentrations of ethanol and Embedded in tacrylate and low speed bone saw (Isomet, Buehler, Lake Bluff, IL) ) To cut to a thickness of about 100 μm. Cross section cut from the central axis of the base of the femur (2-3 ) Is adhered to a plastic slide, and the ground and Polish and cortical internal structure were analyzed under polarized light. Measurements are taken internally and The thickness of the outer lamellae bone layer and the thickness of the intermediate reticulated bone were included. After these measurements The samples were stained by the von Kossa method, counterstained with toluidine blue, and Burslip-covered, static bone analysis was performed, including a second inertial parameter The calculation of the moment was included.                             4. Bone cell analysis   The right tibia was fixed with 10% neutral buffered formalin for 48 hours, and Cal-Rite decalcified solution ( Richland-Allan Medical, Richland, MI), demineralized for 2 weeks, and Processed for paraffin embedding. Long axis cut at 3μm thickness along the proximal intertibia Pieces were prepared using hematoxylin and eosin and gomori trichrome. Stained and used for bone cell analysis.                              5. Statistical analysis   The difference between the plasma glucose values at 0 and 2 hours in Table 3 was Were analyzed by T-test. Table 3 shows the variance to compare the differences between the treatment groups. Two-factor analysis was used. Groups shown in Tables 7-16 or Figures 2a-b, 3, 4, 7a-d Differences between were tested for significant differences in one-way analysis of variance. Analysis of variance is flat If there was a significant difference between the averages, this difference was assessed using Dunnat's T-test and multiple comparisons. Is evaluated using the Fisher's Protected Least Significant Difference method (Netter et al. (1982) "Applied Statistics," Allyn and Bacon, Boston). Statistical significance was considered at p <0.05 and results were expressed as mean ± standard error (SE) expressed. C.result   Analysis of the data indicated that ovariectomy and rhIGF-I and rhIGF-I / IGFBP-3 complex Cortical and cortical site-specific changes to body treatment were demonstrated. Shown in Table 3 Thus, the 2.5 mg / kg dose of rhIGF-I was produced at any dose of rhIGF-I / IGFBP-3. Caused acute hypoglycemia that did not occur. The data shown in Table 4 is for glycosylated No treatment with any effect on moglobin levels and increasing dose RhIGF-I and rhIGFBP-3 are higher than total IGF-I, rhIGF-I and rhIGFBP-3 Indicates that the Qing level has been led. By combining rhIGF-I with rhIGFBP-3 , These factors are higher than observed in rats treated with rhIGF-I alone Offspring serum values were obtained.   The vertebral BMD data from this study is shown in Table 4A, along with the average of individual changes. Forecast As measured, ovariectomy led to a reduction in spinal cord BMD, which was a 7.5 mg / kg dose of rhI. It was prevented by GF-I / IGFBP-3. Some with two low volume rhIGF-I / IGFBP-3 Was observed, but not with the two doses of rhIGF-I. Excised DEXA data obtained for tibia (Table 5) and femur (Table 6) Dose-related in total BMC and total BMD for both and rhIGF-I / IGFBP-3 Indicate an increase.   1.Cortical bone   Treatment with both formulations tested reduces body weight and lean body mass of treated rats. An increase occurred in a dose-dependent manner (FIGS. 2A and 2B). Periosteal osteogenic activity is dependent on all treatments In groups and at all sites measured against sham or OVX controls (Tables 7, 8, 9, and 11). However, 7.5 mg / kg rhIGF-I / IGFBP-3 complex Group of rats treated with the highest increase in periosteal bone formation rate parameters (FIGS. 4A and 5C, 5CC).   Endocortical bone resorption in treated rats (this is estrogen deficient Is a hallmark of cortical bone changes in women and rats) And the values obtained for the tools (FIG. 4B). This is the femoral neck position Shows significantly lower values compared to OVX rats (Table 11). In the endothelial envelope, Bone formation was increased at both bone sites measured (femoral shaft, Table 8).   Internal structure analysis revealed the correctness of the bone formed on the bicortical envelope after rhIGF-I / IGFBP-3 treatment. A normal “lamella” structure was shown (Table 9; FIG. 6). Periosteal and endothelial bone capsule Modeling-dependent increase in bone formation and bone resorption in endothelial bone capsule Relaxation or reduction resulted in thicker cortical bone in treated rats (Table 7). , 9, 11; FIGS. 5 and 6). Increased cortical thickness and newly formed lamellar bone It is mechanically superior to reticular bone), resulting in a 7.5 mg / kg rhIGF-I / IGFBP-3 complex. In treated rats, higher failure torque and polar moment of inertia parameters were observed. A meter was generated (Table 10).   All treated animals have longitudinal bone as compared to OVX or sham rats. It showed a similar increase in growth (FIG. 3). Generally, treatment with rhIGF-I / IGFBP-3 Caused cortical bone thickening by adding “lamellar” bone on both envelopes. Increased fat-free body components increase periosteal bone shape by increasing muscle traction on the periosteum Could be enhanced. Both “lamella” structure and increased cortical thickness are due to rhIGF-I / IGFBP-3 treatment The cortical bone strength after placement was improved.   2.Spongy bone   Examination of spongy bone mechanics and structure at four different locations in the rat skeleton Was. Differences in bone turnover rates at different locations and at indicated locations (distal femur Functional anatomy between the ends, distal femoral metaphysis, femoral midneck, and lumbar vertebrae) Was. Treatment with rhIGF-I / IGFBP-3 increases osteogenic parameters at all four sites (Tables 12, 13, 14, 15; FIGS. 7 and 8). Increased bone formation rate (volume based; FIG. 7) A-7D) are highest in the group treated with 7.5 mg / kg rhIGF-I / IGFBP-3 complex Was. Bone resorption had similar or lower values compared to OVX rats (Tables 12-1 Five). By such a turnover activity, all of the measured in treated rats Increased trabecular thickness and number of trabeculae at sites (excluding femoral metaphysis) .   Primary and secondary spongiosa (spongiosa) at the metaphyseal site of the femur, especially in OVX rats And, after treatment with rhIBF or rhIBF-I / IBFBP-3, very high (FIGS. 9A-9C). This bone site has the highest bone turnover rate You. OVX abolishes “metabolized” trabeculae in the central metaphyseal region with zero mechanical value However, treatment with the rhIGF-I / IGFBP-3 complex helped recovery (FIG. 9C). .   Structural trabecular bone analysis was performed at the femoral neck position. This femoral neck position is The most relevant bone sites (Table 16, FIGS. 10A-10C). Very unique anatomical structure Apart from having bone, this bone site consists of cortical and cancellous bone interconnected by endothelial surfaces. It has both. Cortical bone thickness, spongy bone mass, structure, and endothelial surface and trabecular meshwork Is all important determinant of bone strength in the femoral neck region. rhIG Treatment with F-I / IGFBP-3 increases the interaction between trabecular bone structures and trabecular meshwork and endothelial surfaces. Improved connectivity. In general, the data presented here indicate that a previously ovariectomized rat Reveals the benefits of systemic rhIBF-I / IBFBP-3 for the whole musculoskeletal system became.                                 Example 3                             IGF-I Recombinant synthesis of   A recombinant rhIGF-I was inserted into a gene insert encoding the sequence shown in FIG. Was prepared as described in detail below.material   The bacterial strain used is lysogenized with DE3Escherichia coli K-12 W3110 shares (Studier, F. and Moffat, B, [1986]J. Mol. Biol. 189: 113-130) . This lysogen has a T7 RNA polymerase gene under the control of the lacUV5 promoter. I do.   This host strain was transformed with plasmid pER10088 and selected for tetracycline resistance. did.Plasmid description   The three expression vectors used in this study are inserted downstream of the translation coupler Except that the gene is ubiquitin-IGF, pJU1002 and pJU1003 (Squires, C.H. Et al., [1988]J. Biol. Chem. 263: 16297-16302) (pER10088). further , PER10088 is the plasmidtetSynthetic 16bp adapter sequence at 5 'end of gene Is different from pJU1003 in that it does not include However, the only Pvu in the skeleton derived from pBR322 Has a DNA insert at the II site. pER10088 is the linker 5 '... CCTCGAGG ... 3 at that position 'including.Description of the gene insert   Vectors provided as provided for work performed to support the invention pER10088 is an ATG triplet (initiating), yeast ubiquitin (in 5 'to 3' order) 76 codons, 70 synthetic codons of mature human insulin growth factor I (FIG. 11), And an open reading frame (ORF) consisting of a stop codon . In this case, the ORF was used for fibroblast cell generation as described by Squires et al. (Supra). It is precisely located for the long factor translation coupler.IGF Purification   Produce IGF-IE.coliKeep cells at a temperature below 10 ° C while maintaining the Alternatively, using a microfluidizer, 5 volumes / wt (eg, For example, 1 L / 200 g cell paste) in 50 mM sodium acetate and 1 mM EDTA (pH 5.5) Crushed. Lysates were not disrupted using light microscopyE.coliCell presence Was examined. Add 100 volumes of 10% (w / v) polyethyleneimine solution to the lysate, then add And centrifuged at 10,000 for 20 minutes, and the supernatant was separated from the pellet. 2.5 volumes of pellets Re-extract two more times with 20 mM potassium phosphate, 20 mM DTT and 2 M urea (pH 5.8) And centrifuged as above. Use this pellet for further purification did.   IGF was purified from 5 vol / wt of 20 mM Tris containing 6 M urea, 40 mM DTT and 1 mM EDTA. Extract from the pellet (pH 8.0) and use a Sartorius 0.8μ Filtered through a filter. The filtrate was diluted with the same buffer to a final protein concentration of 3 mg / ml. And then diluted with 2 volumes of 20 mM Tris and 1 mM EDTA (pH 8.0). This Of protamine sulfate and Q-Sepharose according to standard procedures. And subjected to a DNA removal step. Add ubiquitin protein peptidase to the mixture To release IGF-I from the ubiquitin IGF-I fusion protein and allow IGF-I to reach room temperature. For overnight. A typical regeneration reaction is a 20 mM to 50 mM DTT / cystamine ratio. In the presence of Tris buffer 1 and a protein concentration of about 1 mg / ml, 1.5 M to 2 M urea and And pH range 8-9. By the regeneration performed as described above, the initial state of IGF-I About 40% of the correctly regenerated IGF-I compared to the amount resulted.   The regenerated IGF-I solution was clarified, the buffer was changed using an ultrafiltration system, and 50 mM Load on cation exchange column equilibrated in sodium acetate buffer (pH 5.5) did. After loading the IGF-I solution onto the column, the column is washed with equilibration buffer, Then, starting with equilibration buffer, either 0.25M or 0.5M sodium chloride Purified IGF-I was eluted from the column applying a 20 column volume gradient ending in. pure Appropriate fractions of different IGF-I were pooled.   Pool the fractions from the cation exchange column with 10% trifluoroacetic acid (TFA) Acidified to H2.5. The solution is filtered through a 0.2 μm filter and 0.1% TF Loaded on a Vydac C-4 column equilibrated in A aqueous solution. Column with 0.1% TFA It was developed using a linear gradient from 0% to 40% acetonitrile in the presence. Including purified IGF The fractions that had were assayed by SDS-PAGE, then pooled and lyophilized.                                 Example 4                            IGFBP-3 Recombinant synthesis of   Insertion of the recombinant rhIGFBP-3 into the gene encoding the sequence shown in FIG. And made as described in detail below. FIGS. 13-15 Used in the present invention according to standard methods or methods similar to those described below. Figure 4 shows another exemplary gene sequence to be obtained. FIG. 13 is used in the examples herein. It is a preferred coding sequence.material   The bacterial strain used in the experiments performed to support the present invention had DE3 lysogenizedE scherichia  coli It is a derivative of K-12 W3110 strain. (Studier, F. and Moffat, B. [ 1986]J. Mol. Biol. 189: 113-130). This lysogen is under the control of the lacUV5 promoter. With the T7 RNA polymerase gene. Transform this host strain with plasmid pDJ12833. And selected for tetracycline resistance.Plasmid description   The three expression vectors used in this study were those whose gene was the translation coupler IGFBP-3. PJU1002 and pJU1003 except that they were inserted downstream (Squires, C.H. et al., [1988]J. Biol. Chem.  263: 16297-16302, incorporated herein by reference). (PDJ12833). In addition, pDJ12833 istetMatches the 5 'end of the gene It differs from pJU1003 in that it does not contain a 16 bp adapter sequence. However, derived from pBR322 Has a DNA insert at the unique PvuII site in the backbone. pDJ12833 is the pSC101par Includes a 385 bp fragment with a locus (Meacock, P.A. and Cohen, S.N. [1980]C ell  20: 529-542).Description of the gene insert   pDJ12833 is derived from the ATG triplet, followed by 264 codons of mature human IGFBP-3 ORF (Figure 13). The 95 amino-terminal codons were synthesized; It was derived from the natural cDNA for the gene.   In this case, the ORF is based on Squires et al. ([1988]J. Biol. Chem. 263: 16297-16302, this specification Of fibroblast growth factor as described by It is located exactly against the translation coupler.IGFBP-3 Purification   Produces IGFBP-3 ("BP-3")E.coliKeep cells at a temperature below 10 ° C Et al., Using a Gaulin mill or Microfluidizer, 6 volumes / wt (eg, (1.2 l / 200 g cell paste) in 20 mM Tris-HCl and 5 mM EDTA (pH 8) . Lysates were not disrupted using light microscopyE.coliCheck for the presence of cells, then Then, the mixture was centrifuged at 10,000 g for 20 minutes, and the supernatant was separated from the pellet. 3 pellets Re-extract twice more in a volume of 20 mM Tris-HCl (pH 8) and centrifuge as above. Heart isolated. The resulting pellet was used for further purification.   BP-3 was prepared at 2.5 vol / wt 20 mM containing 6 M GdHCl, 25 mM DTT and 5 mM EDTA. Extract from pellet with Tris (pH 8.0) and pass through Sartorius 0.8μ filter And filtered. Dilute the filtrate with the above buffer to a final protein concentration of 4 mg / ml, It was then diluted with one volume of 20 mM Tris and 5 mM EDTA (pH 8.0). This mixture is DNA removal using protamine sulfate according to standard techniques known in the art Subjected to the process. The protamine sulfate precipitate was removed by filtration and BP -3 was subjected to a regeneration reaction. A typical regeneration reaction has a reducing agent / oxidizing agent molar ratio of 1 In the presence of 20 mM to 50 mM Tris buffer, a protein concentration of about 0.5 mg / ml to 1 mg / ml, 1.0 Performed with M-1.5M GdHCl and pH 8-9. With the regeneration performed as described above, Over 60% of the initial amount of BP-3 is produced.   Clarify the regenerated BP-3 solution, replace the buffer using an ultrafiltration system, and Loaded on a cation exchange column equilibrated in sodium acid buffer (pH 7) . After loading the BP-3 regeneration solution onto the cation exchange column, equilibrate the column with buffer And then purify the purified BP-3 with 0.8 M sodium chloride starting with equilibration buffer. The column was eluted by applying a gradient of over 20 column volumes. Appropriate fraction of BP-3 Was pooled.   Increase salt concentration of pooled BP-3 containing fractions to 0.6M using ammonium sulfate And then equilibrated with a salt containing acetate / phosphate buffer having a pH between 5 and 6 Loaded on a hydrophobic interaction chromatography matrix column. BP-3 Linear ammonium sulfate gradient ending with 0% ammonium sulfate buffered at pH 5-6 And eluted. The appropriate fractions were then pooled and saved.   The pooled fractions containing BP-3 were taken up in 0.1% trifluoroacetic acid (TFA) and The solution was then loaded onto a C4 RP-HPLC column equilibrated with 0.1% aqueous TFA. Mosquito The ram is washed with equilibration buffer and then a linear gradient of acetonitrile containing 0.1% TFA. And eluted. Fractions containing BP-3 were pooled and lyophilized.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/02 C12P 21/02 C (C12P 21/02 A61K 37/24 C12R 1:19) C12N 15/00 A C12N 15/09 C12P 21/02 (C12P 21/02 C12R 1:19) (72) Inventor Bromerge, Robert United States of America 95051, Santa Clara, Flora Vista Avenue No. 508 3775 (72) Inventor Rosen, David M. United States of America 95135, San Jose, Little Worth Way 4142 (72) Inventor Adams, Stephen W. California 94087, Sunnyvale, Inverness Way 914

Claims (1)

[Claims] 1. To stimulate bone formation in subjects with bone marrow disease causing bone loss Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3. The process. 2. The bone marrow disease is plasma cell disorder, leukemia, lymphoma, systemic mastocytosis, poor 2. The composition of claim 1, wherein the composition is selected from the group consisting of blood, lipidosis, and mucopolysaccharidosis. the method of. 3. 2. The method of claim 1, wherein a pharmaceutically effective dose of an inhibitor of bone resorption is also administered. the method of. 4. The IGF-I / IGFBP and the inhibitor of bone resorption are administered sequentially, The method of claim 3. 5. The bone resorption inhibitors are estrogen, tamoxifen, calcitonin 4. The method according to claim 3, which is a bisphosphonate or a growth factor having anti-absorption activity. the method of. 6. The method according to claim 5, wherein the growth factor is transforming growth factor β. Method. 7. 4. The method of claim 3, wherein said treatment is prophylactic. 8. Stimulates bone formation in subjects with connective tissue disease causing bone loss Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3. A method comprising the steps of: 9. Wherein the connective tissue disease is osteogenesis imperfecta, Ehlers-Sudanlos syndrome, Malf From Hwang syndrome, cutaneous laxity, homocystinuria, Mankes syndrome and scurvy 9. The method of claim 8, wherein the method is selected from the group consisting of: 10. 10. The method of claim 9, wherein a pharmaceutically effective dose of a bone resorption inhibitor is also administered. The method described. 11. Method for stimulating bone formation in a subject with drug-related osteoporosis Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3. how to. 12. The drug causing osteoporosis is corticosteroid, heparin, oral Anticoagulants, anticonvulsants, methotrexate, thyroid hormone, lithium and Gonna 12. The method of claim 11, wherein the method is selected from the group consisting of dotropin-releasing analogs. . 13. 12. The method of claim 11, wherein a pharmaceutically effective dose of an inhibitor of bone resorption is also administered. The described method. 14． Pregnancy, lactation, chronic hypophosphatemia, hyperphosphatasia, insulin-dependent sugars Urine disease, anorexia nervosa, cadmium poisoning, juvenile osteoporosis or paget of bone A method for stimulating bone formation in a subject having bone loss due to disease. Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3 ,Method. 15. 15. The method of claim 14, wherein a pharmaceutically effective dose of an inhibitor of bone resorption is also administered. The described method. 16. A method for stimulating bone formation in a subject having periodontal bone loss, the method comprising: Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3. One Law. 17． For stimulating bone formation in a subject with osteoarthritis-related bone loss Administering to the subject a pharmaceutically effective dose of IGF-I and IGFBP-3 A method comprising: 18. A composition for inducing osteogenesis in a subject, comprising a pharmaceutically acceptable composition. A composition comprising IGF-I, IGFBP and an inhibitor of bone resorption in a functional excipient. 19. Inhibitors of the bone resorption are estrogen, tamoxifen, calcito 19. A nin, a bisphosphonate or a growth factor having anti-absorption activity. A composition according to claim 1. 20. The IGF-I or IGFBP is conjugated to an inhibitor of bone resorption. 19. The composition according to 18. 21. 19. The composition of claim 18, further comprising a sustained release vehicle. 22. 19. The composition according to claim 18, wherein said IGFBP is IGFBP-3.